Na+-independent nucleoside transport systems are facilitated-diffusion systems, and two distinct classes have been identified according to their sensitivity to the inhibitor, nitrovenzylthioinosine (NBMPR). Both NBMPR- sensitive [ENT1 designated the cloned transporter; ES designation refers to physiological studies (Equilibrative NBMPR-Insensitive) systems are of broad selectivity, transporting both ubiquitously expressed, whereas the EI system has been described only in intestine, leukemia cells and cardiovascular tissues/cells. Both isoforms appear to be involved in scavenging nucleosides, which is especially important in cells that are unable to synthesize nucleosides de novo, such as those of the intestinal epithelium. Adenosine, a substrate of both the ES and EI nucleoside transport systems, is released during ischemic episodes and stimulates chloride secretion. Activation of pro-secretory adenosine A2b receptors may occur when normal basolateral nucleoside uptake mechanisms (scavenging mechanisms) are overwhelmed and local concentrations of adenosine increase, a response that may contribute to the diarrheal response associated with mesenteric ischemia. In T84 cells, a model for intestinal chloride- secreting crypt cells, Na-independent nucleoside transporters on the basolateral membranes have been synthesized to help in maintaining extracellular concentrations of adenosine below the threshold of activation of the adenosine A2b with respect to nucleoside transport and protein kinase regulation in a transporter deficient cell line, PK-15NTD. In addition, structure/function studies re proposed to understand how the ENT1 transport functions at the molecular level, including the role of glycosylation, the role of cystein residues in regulating function, and whether the transporter functions as a monomer or an oligomer. To address the physiological roles of ENT1 and ENT2 in regulating intestinal adenosine-mediated chloride secretion, T84 cells will be mutated to obtain cells lines that express only ENT1, or cell lines deficient in both ENT1 and ENT2. In short-circuit current experiments, wild-type and mutant cell lines will then be studied to elucidate the contribution of both transporters to the regulation of chloride secretion. Our proposed studies should provide insights into the molecular mechanisms and regulation of Na+-independent nucleoside transporters, as well as their role in scavenging adenosine in inflammatory bowel disease and certain infectious causes of diarrhea.
